The present invention relates to an isolated linoleate isomerase enzyme, an nucleic acid molecule encoding a linoleate isomerase enzyme, to immobilized cells containing a linoleate isomerase enzyme, and to a method for converting linoleic acid or linolenic acid to CLA using the isolated linoleate isomerase enzyme, nucleic acid molecule and/or immobilized cells.
The term xe2x80x9cCLAxe2x80x9d is used herein as a generic term to describe both conjugated linoleic acid and conjugated linolenic acid. The CLA compounds (cis,trans)-9,11-linoleic acid and (trans,cis)-10,12-linoleic acid are recognized nutritional supplements and effective inhibitors of epidermal carcinogenesis and forestomach neoplasia in mice, and of carcinogen-induced rat mammary tumors. CLA has also been shown to prevent adverse effects caused by immune stimulation in chicks, mice and rats, and has been shown to decrease the ratio of low density lipoprotein cholesterol to high density lipoprotein cholesterol in rabbits fed an atherogenic diet. CLA also reduces body fat in mouse, rat, chick and pig models. CLA has also been shown to be effective in treating skin. lesions when included in the diet.
CLA occurs naturally in various amounts in virtually all foods. The principle natural sources of CLA are dairy products, beef and foods derived from ruminant animals. In the U.S., beef, beef tallow, veal, lamb (3-4 mg CLA/g fat; 84% cis-9, trans-11) and dairy products (3-7 mg CLA/g fat; 80-90% cis-9, trans-11) have the highest concentration of CLA. CLA concentrations 2-3 times higher are found in Australian dairy products and pasture-fed beef and lamb. Very low concentrations of CLA (0.1-0.7 mg CLA/g fat; ca. 40% each cis-9, trans-11 and trans-10, cis-12) are found in commercial vegetable oils.
CLA is a normal intermediate of linoleic acid metabolism. In cows, (cis,trans)-9,11-CLA produced by natural bacterial flora that is not further metabolized is incorporated into lipids and then into host tissues and milk. Animals take up and incorporate CLA into normal tissue and milk from dietary sources such as milk, milk products or meat containing CLA, or from CLA dietary supplements.
CLA can be synthetically obtained from alkaline isomerization of linoleic or linolenic acid, or of vegetable oils which contain linoleic acid, linolenic acid or their derivatives. Heating vegetable oil at about 180xc2x0 C. under alkaline conditions catalyzes two reactions: (1) fatty acid ester bonds from the triglyceride lipid backbone are hydrolyzed, producing free fatty acids; and (2) unconjugated unsaturated fatty acids with two or more appropriate double bonds are conjugated. Commercial CLA oils available at the present time, typically made from sunflower oil, are sold without further purification. They contain a mixture of CLA isomers as well as other saturated and unsaturated fatty acids. Generally, chemical synthesis produces about 20-35% (cis,trans)-9,11-CLA and about 20-35% (trans,cis)-10,12-CLA, and the balance as a variety of other isomers. The presence of the non-active, non-natural isomers introduces the need to purify (cis,trans)-9,11-CLA and/or (trans,cis)-10,12-CLA, or to demonstrate the safety and seek regulatory approval of these non-beneficial, non-natural isomers for human use. It is not feasible economically, however, to isolate single isomers of CLA from the CLA made by alkaline isomerization. Using a fractional crystallization procedure, it is possible to enrich 9,11-CLA relative to 10,12-CLA and vice versa. Another approach, described in WO 97/18320 to Loders Croklaan B. V. uses lipases to selectively esterify 10,12-CLA and thus enrich the 9,11-CLA fraction. None of the above-described methods, however, allow for the production of high purity, single isomer CLA.
One method of overcoming the shortcomings of chemical transformation is a whole cell transformation or an enzymatic transformation of linoleic acid, linolenic acid or their derivatives to CLA. It is well known that a biological system can be an effective alternative to chemical synthesis in producing a desired chemical compound where such a biological system is available. The existence of linoleate isomerase enzyme to convert linoleic acid to CLA has been known for over thirty years, however, no one has yet successfully isolated the enzyme. And because it has not yet been isolated, the linoleate isomerase enzyme has not been sequenced.
In many microorganisms, the linoleate isomerase enzyme converts linoleic acid to CLA as an intermediate in the biohydrogenation step. Kepler and Tove have identified this enzyme in Butyrivibrio fibrisolvens. Kepler and Tove, J. Biol. Chem., 1966, 241, 1350. However, they could not solubilize the activity, i.e., they were unable to isolate the enzyme in any significantly pure form. Kepler and Tove, J. Biol. Chem., 1967, 242, 5686. In addition, earlier studies have indicated that only compounds which possess a free carboxyl group and a cis-9, cis-12 double bond moieties are isomerized by linoleate isomerase. See Kepler and Tove, Methods in Enzymology, 1969, 14, 105-109, and Kepler et al., J. Biol. Chem., 1970, 245, 3612.
Therefore, there remains a need for purifying and identifying a linoleate isomerase enzyme and/or producing one by recombinant techniques. There also remains a need for finding and identifying an linoleate isomerase enzyme which does not require presence of a free carboxylic acid group in the fatty acid for isomerization. In addition, there remains a need for a method for producing CLA utilizing whole cells or isolated linoleate isomerase enzyme.
The present invention generally relates to isolated linoleate isomerase nucleic acid molecules, isolated linoleate isomerase proteins, immobilized bacterial cells having a genetic modification that increases the action of linoleate isomerase, and methods of using such nucleic acid molecules, proteins and cells to produce CLA.
One embodiment of the invention relates to an isolated linoleate isomerase. Included in the invention are linoleate isomerases from Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio and Eubacterium, and particularly, from Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Butyrivibrio fibrisolvens, Propionibacterium acidipropionici, Propionibacterium freudenreichii and Eubacterium lentum. Particularly preferred linoleate isomerases include linoleate isomerases from Lactobacillus reuteri, Clostridium sporogenes, and Propionibacterium acnes. 
In one embodiment, an isolated linoleate isomerase of the present invention converts linoleic acid and linolenic acid to CLA, including (cis, trans)-9,11-linoleic acid and/or (trans, cis)-10,12-linoleic acid. A linoleate isomerase of the present invention includes linoleate isomerases having one or more of the following biochemical characteristics: a size of about 50 kDa or about 67 kDa; an optimum pH of about 6.8 or about 7.5; a specific linoleic acid isomerization activity of at least about 1000 nmoles mgxe2x88x921 minxe2x88x921; a Km of about 8.1 xcexcM for linoleic acid, a pH optimum of about 7.5, and a Ki of about 80 xcexcM for oleic acid; and/or an initial velocity that decreases at about 60 xcexcM linoleic acid. A linoleate isomerase of the present invention can be either a membrane bound or a soluble enzyme. The linoleate isomerase of the present invention can include lipid material.
In another embodiment, an isolated linoleate isomerase of the present invention includes an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the complement of a sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26. In another embodiment, the isolated linoleate isomerase is encoded by a nucleic acid molecule which includes a nucleic acid sequence having at least 24 contiguous nucleotides having 100% identity with nucleic acid sequence SEQ ID NO:17. In yet another embodiment, an isolated linoleate isomerase of the present invention includes an amino acid sequence with at least about 70% identity with an amino acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:18.
Preferably, an isolated linoleate isomerase is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26, with SEQ ID NO:17 being the most preferred. Isolated linoleate isomerases of the present invention include proteins having an amino acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:18, with SEQ ID NO:18 being most preferred. Also included in the present invention are homologues of linoleate isomerase proteins, including proteins having an amino acid sequence having at least 8 contiguous amino acids with 100% identity to SEQ ID NO:18, and proteins encoded by naturally occurring allelic variants of linoleate isomerase nucleic acid molecules.
In one embodiment, the linoleate isomerase is bound to a solid support, which includes, but is not limited to organic supports, biopolymer supports and inorganic supports.
Another embodiment of the present invention relates to an isolated antibody that selectively binds to the isolated linoleate isomerase of the present invention.
Yet another embodiment of the present invention relates to a method for producing CLA, including contacting an oil, which comprises a compound selected from the group of linoleic acid and linolenic acid, with an isolated linoleate isomerase enzyme of the present invention to convert at least a portion of the compound to CLA. In one embodiment, the compound is in the form of a triglyceride and the method further includes contacting the oil with a hydrolysis enzyme to convert at least a portion of the triglyceride to free fatty acids. Such a hydrolysis enzyme can include lipases, phospholipases and esterases. The method of the present invention can also include a step of recovering the CLA. The CLA can included (cis, trans)-9,11-linoleic acid and/or (trans, cis)-10,12-linoleic acid. The oil can include, but is not limited to, sunflower oil, safflower oil, corn oil, linseed oil, palm oil, rapeseed oil, sardine oil, herring oil, mustard seed oil, peanut oil, sesame oil, perilla oil, cottonseed oil, soybean oil, dehydrated castor oil and walnut oil. In one embodiment of the method, the linoleate isomerase enzyme is bound to a solid support, which can include organic supports, biopolymer supports and inorganic supports.
Another aspect of the present invention relates to an isolated nucleic acid molecule selected from the group of: (a) a nucleic acid molecule comprising a nucleic acid sequence that encodes a protein having an amino acid sequence selected from the group of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:18; (b) a nucleic acid molecule encoding a homologue of any of such amino acid sequences of (a), wherein the homologue comprises at least 8 contiguous amino acids having 100% identity with amino acids in such amino acid sequences; (c) a nucleic acid molecule comprising a naturally occurring allelic variant of a nucleic acid molecule encoding any of such amino acid sequences of (a);
and, (d) a nucleic acid molecule that is complementary to any of the nucleic acid molecules of (a), (b) or (c). Preferably, an isolated nucleic acid molecule of the present invention encodes a linoleate isomerase, including a linoleate isomerase homologue. In one embodiment, such an isolated nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:26, and/or the complement of any of such nucleic acid sequences. In another embodiment, an isolated nucleic acid molecule of the present invention includes a nucleic acid sequence having at least about 70% identity with a nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26. In yet another embodiment, an isolated nucleic acid molecule of the present invention includes a nucleic acid sequence having at least 24 contiguous nucleotides having 100% identity with nucleic acid sequence SEQ ID NO:17. Preferred nucleic acid molecules of the present invention include molecules that hybridize under stringent hybridization conditions with a nucleic acid molecule selected from the group of nCLA87, nCLA596 nCLA1709, nCLA3551, nCLA1776 and nCLA7113. More preferably, an isolated nucleic acid molecule of the present invention has a sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:26, with SEQ ID NO:17 being more preferred. An isolated nucleic acid molecule of the present invention preferably encodes a linoleate isomerase protein of the present invention as described above.
The isolate nucleic acid molecule of the present invention includes linoleate isomerase nucleic acid molecules from microorganisms including, but not limited to, Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio, and Eubacterium, with Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Butyrivibrio fibrisolvens, Propionibacterium acidipropionici, Propionibacterium freudenreichii and Eubacterium lentum being particularly preferred. Most preferred linoleate isomerase nucleic acid molecules are from Lactobacillus reuteri, Clostridium sporogenes, or Propionibacterium acnes. 
Also included in the present invention are recombinant molecules, recombinant viruses and recombinant cells which include an isolated nucleic acid molecule of the present invention. In one embodiment, as recombinant cell of the present invention is from a microorganism which includes, but is not limited to, Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Butyrivibrio fibrisolvens, or Eubacterium lentum, with Escherichia coli, Bacillus subtilis and Bacillus licheniformis being most preferred.
Yet another embodiment of the present invention relates to a method to produce linoleate isomerase, comprising culturing a recombinant cell transformed with an isolated nucleic acid molecule encoding linoleate isomerase.
Another embodiment of the present invention relates to a method for producing CLA, including contacting an oil which comprises a compound selected from the group of linoleic acid and linolenic acid, with an isolated linoleate isomerase enzyme encoded by the isolated nucleic acid molecule of the present invention to convert at least a portion of the compound to CLA.
Yet another embodiment of the present invention relates to an immobilized bacterial cell having a genetic modification that increases the action of linoleate isomerase. The cell can be a microorganism which includes, but is not limited to Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio, Escherichia, Bacillus or Eubacterium cells. In one embodiment, the genetic modification results in overexpression of linoleate isomerase by the bacterial cell. The genetic modification can result in at least one amino acid modification selected from the group consisting of deletion, insertion, inversion, substitution and derivatization of at least one amino acid residue of the linoleate isomerase, wherein such modification results in increased linoleate isomerase action, reduced substrate inhibition, and/or reduced product inhibition. In another embodiment, the genetic modification includes transformation of the cell with a recombinant nucleic acid molecule encoding a linoleate isomerase of the present invention, wherein the recombinant nucleic acid molecule is operatively linked to a transcription control sequence. The recombinant nucleic acid molecule can include any of the isolated nucleic acid molecules described above, including a nucleic acid sequence encoding a homologue of linoleate isomerase. In one embodiment, such a homologue has an amino acid sequence having at least 8 contiguous amino acids with 100% identity to amino acid sequence SEQ ID NO:18.
In one embodiment, the recombinant nucleic acid molecule is integrated into the genome of the bacterial cell. In another embodiment, the recombinant nucleic acid molecule encoding linoleate isomerase comprises a genetic modification which increases the action of the linoleate isomerase and in another embodiment, the genetic modification reduces substrate and/or product inhibition of the linoleate isomerase.
In another embodiment, an immobilized bacterial cell of the present invention can be lysed. The cell can be immobilized by crosslinking with a bifunctional or multifunctional crosslinking agent, including, but not limited to glutaraldehyde.
Yet another embodiment of the present invention relates to a method for producing CLA, including contacting an oil which includes a compound selected from the group of linoleic acid and linolenic acid, with an immobilized bacterial cell having a linoleate isomerase, to convert at least a portion of the compound to CLA. The bacterial cell can be from a microorganism including Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio, Escherichia, Bacillus and Eubacterium cells, preferably Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Butyrivibrio fibrisolvens, and Eubacterium lentum, and most preferably, Escherichia coli, Bacillus subtilis or Bacillus licheniformis. The cell can be a naturally occurring bacterial cell having a linoleate isomerase, or a genetically modified microorganism as described above. Preferably, a genetically modified microorganism has increased linoleate isomerase action. The compound can include compounds in the form of a triglyceride such that at least a portion of the triglycerides are converted to free fatty acids. Other features of the method are as described above in the method to produce CLA.